Src-family Tyrosine Kinases in Activation of ERK-1 and p85/p110-phosphatidylinositol 3-Kinase by G/CCKBReceptors

Abstract

We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc·Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src·IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin. We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc·Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src·IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin. Gastrin was initially characterized as the major hormonal regulator of gastric acid secretion. This polypeptide also functions as a growth factor on the gastrointestinal tract in vivo (1Johnson L.R. Annu. Rev. Physiol. 1977; 39: 135-158Crossref PubMed Scopus (70) Google Scholar). Its trophic effects have also been observed on several cancer cell lines derived from colon stomach and pancreas (2Kusyk C.J. McNeil N.O. Johnson L.R. Am. J. Physiol. 1986; 251: G597-G601Crossref PubMed Google Scholar, 3Seva C. Scemama J.L. Bastie M.J. Pradayrol L. Vaysse N. Cancer Res. 1990; 50: 5829-5833PubMed Google Scholar, 4Seva C. Dickinson C.J. Yamada T. Science. 1994; 265: 410-412Crossref PubMed Scopus (311) Google Scholar). Gastrin effects have been shown to be mediated by the gastrin/CCKB(G/CCKB) 1The abbreviations G/CCKBgastrin/CCKBERKextracellular-regulated kinaseGPCRG protein-coupled receptorPI 3-kinasephosphatidylinositol 3-kinasePtdInsphosphatidylinositolsIRSinsulin receptor substrateCHOChinese hamster ovaryIPimmunoprecipitationIBimmunoblot 1The abbreviations G/CCKBgastrin/CCKBERKextracellular-regulated kinaseGPCRG protein-coupled receptorPI 3-kinasephosphatidylinositol 3-kinasePtdInsphosphatidylinositolsIRSinsulin receptor substrateCHOChinese hamster ovaryIPimmunoprecipitationIBimmunoblotreceptor, which belongs to the family of G protein-coupled receptors (5Wank S.A. Pisegna J.R. Weerth A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8691-8696Crossref PubMed Scopus (461) Google Scholar, 6Kopin A.S. Lee Y.M. Mc Bride E.W. Miller L.J. Kolakowski L.F. Beinborn M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3605-3610Crossref PubMed Scopus (468) Google Scholar). gastrin/CCKB extracellular-regulated kinase G protein-coupled receptor phosphatidylinositol 3-kinase phosphatidylinositols insulin receptor substrate Chinese hamster ovary immunoprecipitation immunoblot gastrin/CCKB extracellular-regulated kinase G protein-coupled receptor phosphatidylinositol 3-kinase phosphatidylinositols insulin receptor substrate Chinese hamster ovary immunoprecipitation immunoblot Extracellular-regulated kinase-1 and -2 (ERK-1 and ERK-2) are two members of the mitogen-activated protein kinase family known to play an important role in cell proliferation. They have been shown to be activated by a variety of receptors including receptor tyrosine kinases and G protein-coupled receptors (GPCRs). Their activation by growth factor receptors with intrinsic tyrosine kinase activity has been well documented. The best understood mechanism involves the recruitment of the Grb2·Sos or Shc·Grb2·Sos complexes to tyrosine-phosphorylated receptors. These complexes subsequently activate the following cascade: Ras/Raf/ERK kinases/ERKs (7Serger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3175) Google Scholar). Src-family tyrosine kinases have been proposed to serve as intermediates between GPCR and ERK activation. However, their involvement in this transduction pathway seems to be highly receptor- and cell type-specific. In some cells, lysophosphatidic acid and angiotensin receptors can activate ERKs via a mechanism that involves the tyrosine phosphorylation of Shc proteins by Src-family kinases (8Luttrell L.M. Hawes B.E. Van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 9Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar, 10Daub H. Wallasch C. Lankenau A. Herrlich A. Ullrich A. EMBO J. 1997; 16: 7032-7044Crossref PubMed Scopus (584) Google Scholar, 11Zou Y. Komuro I. Yamazaki T. Kudoh S. Aikawa R. Zhu W. Shiojima I. Hiroi Y. Tobe K. Kadowaki T. Yazaki Y. Circ. Res. 1998; 82: 337-345Crossref PubMed Scopus (132) Google Scholar). In other cell types, neither Src nor Shc are required for ERK activation by these ligands (12Kranenburg O. Verlaan I. Hordijk P.L. Moolenaar W.H. EMBO J. 1997; 16: 3097-3105Crossref PubMed Scopus (88) Google Scholar). The mechanism leading to ERK activation by α1-B, α2-A adrenergic receptors, muscarinic acetylcholine, or bradykinin receptors also involves Src and Shc (9Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar, 13Della Rocca G.J. Van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 14Wan Y. Kurosaki T. Huang X.-Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (256) Google Scholar), whereas thrombin or formylmethionylleucylphenylalanine induce the ERK cascade independently of these two proteins (15Chen Y.H. Grall D. Salcini A.E. Pelicci P.G. Pouyssegur J. Van Obberghen-Schilling E. EMBO J. 1996; 15: 1037-1044Crossref PubMed Scopus (104) Google Scholar, 16Torres M. Ye R.D. J. Biol. Chem. 1996; 271: 13244-13249Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). We and others have recently shown that gastrin modulates ERK-1/2 activity; however, the contribution of Src-family kinases and Shc proteins has never been studied (17Taniguchi T. Matsui T. Ito M. Murayama T. Tsukamoto T. Katakami Y. Chiba T. Chihara K. Oncogene. 1994; 9: 861-867PubMed Google Scholar, 18Seva C. Kowalski-Chauvel A. Blanchet J.S. Vaysse N. Pradayrol L. FEBS Lett. 1996; 378: 74-78Crossref PubMed Scopus (44) Google Scholar, 19Seufferlein T. Withers D.J. Broad S. Herget T. Walsh J.H. Rozengurt E. Cell Growth Differ. 1995; 6: 383-393PubMed Google Scholar). Phosphatidylinositol 3-kinases (PI 3-kinases) are lipid kinases that phosphorylate phosphatidylinositols (PtdIns) at the D-3 position of the inositol ring. Several PI 3-kinase isoforms have been described in the literature. The first cloned PI 3-kinase was a heterodimer composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit, which has been implicated in the regulation of mitogenesis and cell transformation (20Escobedo J.A. Navankasattusas S. Kavanaugh W.M. Milfray D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (372) Google Scholar, 21Skolnick E.Y. Margolis B. Mohamnudi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 22Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayoto G. Thompson A. Dhanal R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1992; 65: 91-104Abstract Full Text PDF Scopus (538) Google Scholar). This enzyme has been found to be associated and activated by growth factor receptors (23Coughlin S.R. Escobedo J.A. Williams L.T. Science. 1989; 243: 1191-1194Crossref PubMed Scopus (287) Google Scholar, 24Fantl W.J. Escobedo J.A. Martin G.A. Turck C.W. Del Rosario M. McCormick F. Williams L.T. Cell. 1992; 69: 413-423Abstract Full Text PDF PubMed Scopus (471) Google Scholar). In addition, inhibition of PI 3-kinase activity by specific inhibitors or antibodies results in blockage of growth factor-induced cell proliferation (25Roche S. Koegl M. Courtneidge S.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9185-9189Crossref PubMed Scopus (261) Google Scholar,26Vemuri G.S. Rittenhouse S.E. Biochem. Biophys. Res. Commun. 1994; 202: 1619-1623Crossref PubMed Scopus (31) Google Scholar). Activation of the p85/p110-PI 3-kinase by tyrosine kinase receptors such as platelet-derived growth factor or colony-stimulating factor-1 receptors involves the binding of the SH2 domain (for Src-Homology 2) of p85 to specific phosphotyrosine-containing sequences of the receptors (27Kashishian A. Kazlaukas A. Cooper J.A. EMBO J. 1992; 11: 1373-1382Crossref PubMed Scopus (200) Google Scholar, 28Reedijk M. Liu X. Van Der Geer P. Letwin K. Waterfield M.D. Hunter T. Pawson T. EMBO J. 1992; 11: 1365-1372Crossref PubMed Scopus (177) Google Scholar). In contrast, the interaction of p85 with a tyrosine-phosphorylated intermediate protein, the insulin receptor substrate-1 (IRS-1), represents a mechanism for p85/p110-PI 3-kinase activation by insulin or insulin growth factor-1 (29White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 30Giorgetti S. Ballotti R. Kowalski-Chauvel A. Tartare S. Van Obberghen E. J. Biol. Chem. 1993; 268: 7358-7364Abstract Full Text PDF PubMed Google Scholar). Quite recently, novel forms of PI 3-kinases have been characterized. They do not interact with p85 and are directly activated by GPCRs and βγ subunits of G proteins (31Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (515) Google Scholar, 32Thomason P.A. James S.R. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar, 33Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Science. 1995; 269: 690-693Crossref PubMed Scopus (637) Google Scholar). Several laboratories including ours have reported that some GPCRs such as the G/CCKB are capable of activating the p85/p110-PI 3-kinase (34Hu Z.W. Shi X.Y. Lin R.Z. Hoffman B.B. J. Biol. Chem. 1996; 271: 8977-8982Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 35Saward L. Zahradka P. Circ. Res. 1997; 81: 249-257Crossref PubMed Google Scholar, 36Kowalski-Chauvel A. Pradayrol L. Vaysse N. Seva C. J. Biol. Chem. 1996; 271: 26356-26361Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, very little is known about the mechanism of activation. In this study, we have analyzed the contribution of Src-family tyrosine kinases to the activation of ERK-1 or p85/p110-PI 3-kinase pathways by gastrin. We report here that Shc tyrosine phosphorylation by Src-family tyrosine kinases and Shc·Grb2 complex formation are early events in ERKs activation via the GPCR, G/CCKB. Similarly, Src-family tyrosine kinases, by phosphorylating IRS-1, play an essential role in the gastrin-mediated p85/p110-PI 3-kinase-signaling cascade. Chinese hamster ovary (CHO) cells, stably transfected with an expression plasmid encoding the human G/CCKB receptor and designated as control cells, were grown in α-minimum essential medium supplemented with 10% fetal and at in a The Shc of the SH2 domain of or the encoding were with in cells the human G/CCKB receptor the cell lines of were characterized and with control cells. The was a from I. and P. G. Pelicci The encoding was from S. or Csk-transfected cells were for the cells were and the of were as described A. Pradayrol L. Vaysse N. Seva C. J. Biol. Chem. 1996; 271: 26356-26361Abstract Full Text Full Text PDF PubMed Scopus (65) Google with the from immunoprecipitates or cell were by and to were as described A. Pradayrol L. Vaysse N. Seva C. J. Biol. Chem. 1996; 271: 26356-26361Abstract Full Text Full Text PDF PubMed Scopus (65) Google with the were by by were with and with were at for in of kinase of and as an were analyzed by The were with at for and described in G. S. Koegl M. Courtneidge S.A. G. EMBO J. 1993; PubMed Scopus Google Scholar), with specific antibodies were at for in of and of The was by the on in in the was described C. Kowalski-Chauvel A. Blanchet J.S. Vaysse N. Pradayrol L. FEBS Lett. 1996; 378: 74-78Crossref PubMed Scopus (44) Google Scholar), with specific antibodies were for at in protein and The was as described described A. Pradayrol L. Vaysse N. Seva C. J. Biol. Chem. 1996; 271: 26356-26361Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), the immunoprecipitates were in The kinase was by of and at a of the was by the of and the were with The were by and analyzed by was by of the The was the of the The cells are with medium to was with 10% acid at for were with in of and analyzed in a and were from and and were from (for immunoprecipitation anti-IRS-1 (for immunoblot and (for antibodies were from (for and (for were from and antibodies were from antibodies were from Science. antibodies specific to p85 were by Y. and J. F. antibodies (for were from E. Van Obberghen and I. Shc proteins are for gastrin-mediated ERK we stably transfected in cells the G/CCKB receptor, a inhibitory mutant of Shc of the SH2 domain of Expression of the Shc mutant completely blocked the increase of gastrin-stimulated Shc tyrosine phosphorylation and Shc·Grb2 complex formation of the with the antibodies for immunoprecipitation of protein in precipitates from and cells shown in of the Shc mutant in inhibition of ERK-1 suggesting that and pathways gastrin on ERK activation. Src tyrosine kinase activity has been shown to be highly by tyrosine of the the enzyme in an whereas the to the phosphorylation of the kinase domain S.A. S. Koegl M. G. 1993; Google Scholar). reported the tyrosine phosphorylation of Src-like proteins in response to gastrin P. S. Am. J. Physiol. 1994; Google Scholar, 1998; PubMed Scopus Google Scholar). However, this phosphorylation is inhibitory or to be We first the of gastrin to the kinase activity of Src in cells transfected with the G/CCKB of the cells with the of Src to phosphorylate the was measured in anti-Src immunoprecipitates as described The of Src activation by gastrin was a at and subsequently the role of Src-family tyrosine kinases in the mechanisms activated by we a regulatory protein of Csk, which inactivates Src-family kinases by phosphorylation S.A. S. Koegl M. G. 1993; Google Scholar). Overexpression of in cells completely blocked gastrin-induced Src kinase activity of the for the kinase was with an anti-Src and with the to that of Src were in and Csk-transfected cells the contribution of Src-family kinases to gastrin-induced ERKs we measured ERK-1 activity in cells Csk. of gastrin-induced Src kinase activity by also in a but inhibition of ERK-1 activation by gastrin the effects of on Shc·Grb2 complex activation by gastrin. In control cells, the effects of gastrin on tyrosine phosphorylation of Shc and the between Shc and and of the In Csk-transfected cells, tyrosine phosphorylation of Shc and the between Shc and in response to gastrin were and with control cells, the involvement of Src-family tyrosine kinases for gastrin on Shc·Grb2 complex of the with the antibodies for immunoprecipitation of protein in precipitates from and Csk-transfected cells We Src-like tyrosine kinase activity was detected in with Shc proteins following gastrin kinase activity in Shc immunoprecipitates was measured by as an increase in the kinase activity was detected in anti-Shc precipitates gastrin stimulation and was abolished in cells of proteins of the for the kinase was with an anti-Shc and with the to that of Shc were in and Csk-transfected cells The mechanisms leading to the activation of the p85/p110-PI 3-kinase by GPCRs are activation of in cells the p85/p110-PI PI 3-kinase activity was measured in anti-p85 immunoprecipitates following gastrin We observed a rapid increase in PI 3-kinase activity in response to gastrin. The activation a at and PI 3-kinase activation in anti-p85 immunoprecipitates was completely blocked in Csk-transfected cells suggesting the involvement of Src-like tyrosine kinases in the mechanism of PI 3-kinase activation by gastrin. We not increase in the tyrosine phosphorylation of the p85 subunit of PI 3-kinase in cells not with antibodies from immunoprecipitates with anti-IRS-1 antibodies an tyrosine phosphorylation of IRS-1 following gastrin stimulation In addition, PI 3-kinase activity was detected in with IRS-1 of the cells with of gastrin The of at could be to the rapid phosphorylation of IRS-1, suggesting that the tyrosine phosphorylation of IRS-1, which to the rapid recruitment of the p85/p110-PI be for PI 3-kinase activation by gastrin. the tyrosine phosphorylation of IRS-1 and the PI 3-kinase activity in anti-IRS-1 precipitates observed in response to gastrin were abolished by and suggesting that Src-family tyrosine kinases upstream of the 3-kinase pathway activated by gastrin. We analyzed Src-like tyrosine kinases could IRS-1 in response to gastrin. this cells were with gastrin for and as described IRS-1 proteins were with specific and their with Src kinases was analyzed by with anti-Src increase in the of Src proteins with IRS-1 was detected in response to gastrin In addition, we observed a increase of Src-like kinase activity in anti-IRS-1 immunoprecipitates from cells with gastrin suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src·IRS-1 complex formation in Csk-transfected cells and The of proteins in precipitates from or Csk-transfected cells were by in these with antibodies Gastrin has been shown to be for cells the M. Matsui T. T. Tsukamoto T. Murayama T. N. H. Chiba T. Chihara K. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). we the of Shc proteins and Src-family tyrosine kinases in by this peptide. Overexpression of the Shc mutant or proteins in a inhibition and of the increase in by gastrin that Shc phosphorylation and Src-family kinase activation are two events in growth stimulation by this The mechanisms activated by the are not completely the early events upstream of pathways to be characterized. The important role of Src-family tyrosine kinases in the of by growth receptors intrinsic tyrosine kinase activity is well S.A. S. Koegl M. G. 1993; Google Scholar). In Src platelet-derived growth factor-induced In a cells forms of Src and are to to Overexpression of Src the response to growth factor whereas expression of Src inhibitory the In the study, we demonstrate that a GPCR, and the activity of Src. We also analyzed the and activated by this tyrosine kinase following gastrin The increase in Src-like kinase activity observed in anti-Shc immunoprecipitates from gastrin-stimulated cells that Shc may be a direct substrate of on tyrosine in response to gastrin. This hypothesis was supported by the inhibition of gastrin-induced Shc phosphorylation and Shc·Grb2 complex formation in cells transfected with Csk, known to Src-family tyrosine Our also demonstrate that activation of ERK-1 by gastrin requires Shc phosphorylation by Src-family tyrosine we observed a of gastrin-induced ERK-1 activation in cells or a inhibitory mutant of However, the inhibition in these transfected cells the existence of an pathway for gastrin-mediated ERK-1 stimulation independent of Shc and Src-family tyrosine The protein tyrosine kinase as well as tyrosine which belongs to a family of tyrosine kinases, have also been proposed to GPCRs to the activation of the ERKs cascade (9Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar, Y. K. A. Kurosaki T. A. Huang X.Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). However, seems that these tyrosine kinases interact with Src-family kinases to their activity (9Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (876) Google Scholar, G. Ye D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar, S. J. S.A. S.J. Cell. Biol. 1995; 15: PubMed Scopus Google Scholar). do not to the which the from G/CCKB receptors to the activation of (12Kranenburg O. Verlaan I. Hordijk P.L. Moolenaar W.H. EMBO J. 1997; 16: 3097-3105Crossref PubMed Scopus (88) Google also a regulation of the ERK pathway by GPCRs that do not Src and Shc proteins. Their that a PI activity is independent on Src-family kinases, receptors to ERKs activation. In the study, we demonstrate in cells transfected with the G/CCKB receptor that gastrin-mediated PI 3-kinase activation is totally on Src-family tyrosine PI 3-kinase play a role in the pathway that to ERK-1 activation by gastrin. isoforms of protein kinase are capable of directly activating by phosphorylation M. A. W. Oncogene. 1996; Google Scholar, H. U. I. J. U. Cooper Cell. Biol. 1997; PubMed Scopus Google Scholar). subsequently the ERK kinases, which in activates the This which requires neither the activation of tyrosine kinases nor the formation of the complex may be in the pathway that G/CCKB receptors to ERK-1 activation. several have reported the activation of the of PI 3-kinase by GPCRs including receptors, angiotensin receptors, and adrenergic receptors (34Hu Z.W. Shi X.Y. Lin R.Z. Hoffman B.B. J. Biol. Chem. 1996; 271: 8977-8982Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 35Saward L. Zahradka P. Circ. Res. 1997; 81: 249-257Crossref PubMed Google A. D. Smrcka A. A.E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). However, very little is known about the of this activation. Activation of the enzyme in seems to direct binding of the to the p85 subunit of PI This interaction via the domain (for Src-Homology of and the domain of the p85 subunit as described for the activation of the PI 3-kinase by cell receptor A. D. Smrcka A. A.E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, A. A.E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, W.M. Science. 1994; PubMed Scopus Google Scholar). In the study, we a mechanism for the activation of the p85/p110-PI 3-kinase by Src-family kinases, which IRS-1 as a we demonstrate by that gastrin a rapid tyrosine phosphorylation of IRS-1 as well as the of this protein with Src. We also observed an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of on tyrosine in response to gastrin. This hypothesis was supported by the inhibition of gastrin-induced Src·IRS-1 and IRS-1 phosphorylation in cells transfected with Csk. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation were abolished by The tyrosine phosphorylation of IRS-1 in response to which to has also been observed P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the role of this phosphorylation to be receptors also to the family of They have been shown to the tyrosine phosphorylation of IRS-1 via tyrosine which with receptor and IRS-1 angiotensin in this an inhibition of the PI 3-kinase activity was observed the of the p85 regulatory subunit to IRS-1 F. M.F. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). In contrast, that the tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin. We I. and P. G. Pelicci for the S. for the encoding Csk, Y. and J. F. for antibodies to and E. Van Obberghen and I. for anti-IRS-1